Mannose Targeting and Hydrophobic Tuning of Polycationic Vectors for Efficient Immunostimulatory CpG Delivery

The efficacy of nucleic acid-based therapeutics is often hindered by nuclease degradation and poor cellular uptake. To address these challenges, the complexation with cationic polymers to form polyplexes has been increasingly investigated. In our previous work, we developed a platform technology composed of a mannosylated block for targeting dendritic cells (DCs) via endocytic mannose receptor (CD206), an agmatinyl block for nucleic acid condensation in diblock copolymers (M₁₅-b-A₁₂, M₂₉-b-A₂₅, and M₅₈-b-A₄₅), elongated with a poly-(butyl acrylate) block to promote endosomal escape in triblock copolymers (M₂₉-b-A₂₉-b-B₉ and M₅₈-b-A₅₂-b-B₃₂). We exploited these copolymers to efficiently target DCs for cancer vaccination by delivering plasmid DNA encoding tumor-associated antigens (TAAs), using ovalbumin (pOVA) as a model antigen. However, successful T-cell activation requires an antigen presentation on DCs as major histocompatibility complex (MHC)-antigen complexes, along with immune stimulation, making vaccine adjuvants essential. In this study, we utilized mannosylated cationic copolymers to deliver cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) as a vaccine adjuvant and tested their effect in conjunction with pOVA to further enhance immune activation. Cationic glycopolymers efficiently condensed single-stranded DNA (ssDNA), forming stable, predominantly spherical glycoplexes with sizes ranging from 20 to 40 nm, as assessed by transmission electron microscopy (TEM) analysis. These mannosylated complexes showed high internalization by CD206-expressing cells. Confocal laser microscopy studies revealed rapid nuclear localization mediated by M₅₈-b-A₅₂-b-B₃₂ triblock copolymer and slower endosomal escape for M₅₈-b-A₄₅ diblock copolymer-based glycoplexes. Furthermore, for M₅₈-b-A₄₅ diblock copolymer-based complexes, codelivering CpG and pOVA in the same particles induced stronger DC activation compared to coadministration of glycoplexes containing CpG and glycoplexes containing pOVA. These provide a structure-activity relationship for this class of mannosylated cationic glycopolymers for nucleic acid delivery to DCs and underscore the synergistic benefits of codelivering CpG and nucleic acid encoding TAAs for DC activation.